1. Field of the Invention
The present invention relates to glycosylated prodrugs, to a method of preparing them and to their uses, alone or with tumor-specific immunoezymatic conjugates, particularly in the treatment of cancer.
More specifically, the present invention relates to prodrugs comprising modified anthracyclines which can be cleaved, in particular, by the action of said tumor-specific immunoenzymatic conjugates to give cytotoxic substances which are active towards tumoral cells.
2. Discussion of the Background
The combination of a prodrug with enzyme/monoclonal antibody conjugates as therapeutic agents has been described in the literature. In general, the antibodies in question, which are directed against a specific tissue and are covalently bonded to an enzyme capable of cleaving the prodrug, are first injected into an appropriate animal, especially man, after which a prodrug is administered which can be activated by the enzyme. The prodrug is converted to a cytotoxin by the action of the enzyme/antibody conjugate anchored to the specific tissue, the cytotoxin exerting a cytotoxic effect on said tissue.
International patent application WO 81/01145, in the name of UNIVERSITY OF ILLINOIS FOUNDATION, describes prodrugs which can be activated by hydrolytic enzymes, and defines five criteria for the optimum efficacy of a prodrug: (1) there must be sufficient activating enzyme in the region of the tumor for cytotoxic levels of antitumoral agent to be released in the region of the tumor, (2) the prodrug must not be activated in regions other than that of the tumor, (3) the prodrug must be an appropriate substrate for the enzyme associated with the tumor, under physiological conditions, (4) the prodrug must be non-toxic or much less toxic than the activated antitumoral agent, and (5) the activated substance must have a short biological half-life so that the toxic effects are limited to the tumor.
More specifically, said patent application states that antitumoral agents can be made specific for a tumor by the addition of a peptide converting said agent to a prodrug which is pharmacologically inactive, but can be selectively activated only at the site of the tumor by an enzyme present in large amounts in the region of the tumor (plasmin and plasminogen activator in particular). The amino acid sequence of the peptide part of the prodrug is such that it will be cleaved enzymatically from the antitumoral agent part by proteases such as plasmin or plasminogen activator, so as to release the antitumoral agent in its active form in the region of the tumor.
The prodrugs which can be activated by hydrolytic enzymes can have a structure in which the peptide and the antitumoral part are covalently bonded via a self-sacrificing connector whose molecular structure is such that the enzymatic cleavage of the peptide from the self-sacrificing connector will spontaneously cause the cleavage of its bond with the antitumoral part.
However, the prodrugs described in said International patent application can only be used for cancers which cause an increased production of enzymes, and more particularly proteases, at the site of the tumor; now, these activating enzymes capable of cleaving the prodrugs described in said patent application are not found in sufficient amounts in human cancers, so these prodrugs do not afford the desired selective toxicity (K. D. BAGSHAWE, Br. J. Cancer, 1987, 56, 531).
International patent application WO 88/07378, in the name of CANCER RESEARCH CAMPAIGN TECHNOLOGY LTD, describes a therapeutic system containing on the one hand an enzyme/antibody conjugate and on the other hand a prodrug which can be activated by the enzyme. The antibody of the enzyme/antibody conjugate recognizes a tumor-specific antigen and the enzyme is capable of converting the prodrug to a cytotoxic agent.
Said patent application states that it is preferable to use enzymes other than mammalian enzymes, so as to prevent the premature release of cytotoxic agent by endogenous enzymes.
More specifically, said patent application describes modified nitrogen mustards such as p-bis-N-(2-chloroethyl)aminobenzylglutamic acid and its derivatives) which can be converted to nitrogen mustards in the presence of carboxypeptidases, and anthracyclines in which the terminal amino group is converted to an amide in the presence of an amino acid.
However, these prodrugs have the major disadvantage of retaining considerable intrinsic cytotoxicity.
European patent application 302 473 also describes a therapeutic system which contains two components and in which the enzyme/antibody conjugate located on the tumoral tissue cleaves a prodrug to give a cytotoxic active compound. More specifically, the enzyme/antibody conjugates contain alkaline phosphatase (AP), penicillin V amidase (PVA) or cytosine deaminase (CD) and are used in association with 4'-phosphate etoposide and its derivatives (or 7-(2-aminoethyl phosphate)mitomycin), with N-(p-hydroxyphenoxyacetyl)adriamycin or with 5-fluorocytosine, respectively, as the prodrug.
However, the system described in said patent application has the disadvantage of utilizing either a circulating enzyme, namely alkaline phosphatase, which is capable of activating the prodrug early in the circulation, or an exogenous enzyme (PVA or CD), which is capable of giving rise to intolerance phenomena or sensitization phenomena.
International patent application WO 90/07929, in the name of AKZO NV, describes a site-specific method of activating a prodrug in vivo in an animal by using a conjugate of an activator and a target-specific substance, the activator part of which enables the prodrug to be converted to a pharmacologically active substance. The activator is especially an enzyme of human origin, such as lysozyme, which is absent in the circulation or present in very small amounts, and whose natural substrates are also absent in the circulation or on the surface of the non-target cells. The target-specific substance is especially an antibody directed against a tumor-specific antigen. In particular, the prodrug can comprise an anthracycline (for example doxorubicin) modified by a chitin oligomer bonded to the anthracycline by an amino group at the carbonyl C.sub.13 on the anthracycline or on the glycosylated part.
However, the system proposed in said International patent application has especially the major disadvantage of releasing not doxorubicin itself but a derivative thereof, namely Dox-(GlcNAc).sub.1 or Dox-(GlcNAc).sub.5 ; on the one hand this is not therefore a prodrug in the strict sense, and on the other hand, as far as these derivatives are concerned, there is a lack of accumulated knowledge from both the pharmacological and the toxicological point of view.
It is apparent from the above that the main disadvantages of the systems of the prior art are:
1) as regards the choice of enzyme: PA1 2) as regards the choice of prodrug: PA1 R.sub.4 is a hydrogen atom, a hydroxyl group or a methoxy group; PA1 R is a group CO--CH.sub.2 --R", in which R" is a hydrogen atom, a C.sub.2 -C.sub.6 alkyl group, a hydroxyl group, an alkoxy group, an O-acyl group or an aryl group; PA1 R.sub.5 and R.sub.6, which can be identical or different, are a hydrogen atom or a hydroxyl group; PA1 R.sub.7 is a hydrogen atom or a hydroxyl group; PA1 R.sub.5 is a group --CH.sub.2 --OR.sub.9 or a group COOR.sub.9, where R.sub.9 is a C.sub.1 -C.sub.3 alkyl or a hydrogen atom; PA1 R.sub.10 and R.sub.11 are a hydrogen atom, an acyl protecting group or an alkyl group; PA1 R.sub.12 is a hydroxyl group, an amine group, an amide group or an O-acyl protecting group; PA1 the benzyl --CH.sub.2 is preferably in the para or ortho position to the glycosyl oxygen; and PA1 Y is a hydrogen atom, or at least one electron-attracting group selected especially from the group comprising the NO.sub.2 group, a halogen atom and a group SO.sub.2 X (where X=--CH.sub.3, C.sub.6 H.sub.4 -CH.sub.3, NH.sub.2, N--(C.sub.1 -C.sub.4 alkyl).sub.2 or NH--C.sub.1 -C.sub.4 alkyl), --CN, acyl or COO-alkyl, and/or at least one electron-donating group selected from the group comprising groups of the type O-alkyl, NHCO-alkyl, N(alkyl)CO-alkyl, S-alkyl or alkyl.
the undesired cleavage of the prodrug (circulating enzyme); PA2 the cleavage of the prodrug associated with the production of a large amount of enzyme at the site of the tumor; and PA2 the use of exogenous enzymes capable of giving rise to intolerance phenomena or sensitization phenomena; and PA2 the intrinsic cytotoxicity of the prodrug; PA2 the production of an anthracycline derivative whose pharmacological and toxic effects are not sufficiently well known; and PA2 the use of a prodrug with two compartments (substrate for the enzyme+cytotoxic agent), which has the disadvantage of giving rise to steric or electronic interference with the enzymatic cleavage reaction.
The Applicant consequently set out to provide prodrugs capable of being converted to pharmacologically active substances in the presence of an appropriate enzymatic conjugate, which meet practical needs better than the prodrugs of the prior art, especially in that they are stable, in that they do not give rise to steric or electronic interference during the enzymatic cleavage reaction, and in that they deliver the active cytotoxic substance only at the site of the tumor.